Molecular analysis of the microbial diversity present in the colonic wall, colonic lumen, and cecal lumen of a pig

Abstract

Random clones of 16S ribosomal DNA gene sequences were isolated after PCR amplification with eubacterial primers from total genomic DNA recovered from samples of the colonic lumen, colonic wall, and cecal lumen from a pig. Sequences were also obtained for cultures isolated anaerobically from the same colonic-wall sample. Phylogenetic analysis showed that many sequences were related to those of Lactobacillus or Streptococcus spp. or fell into clusters IX, XIVa, and XI of gram-positive bacteria. In addition, 59% of randomly cloned sequences showed less than 95% similarity to database entries or sequences from cultivated organisms. Cultivation bias is also suggested by the fact that the majority of isolates (54%) recovered from the colon wall by culturing were related to Lactobacillus and Streptococcus, whereas this group accounted for only one-third of the sequence variation for the same sample from random cloning. The remaining cultured isolates were mainly Selenomonas related. A higher proportion of Lactobacillus reuteri-related sequences than of Lactobacillus acidophilus- and Lactobacillus amylovorus-related sequences were present in the colonic-wall sample. Since the majority of bacterial ribosomal sequences recovered from the colon wall are less than 95% related to known organisms, the roles of many of the predominant wall-associated bacteria remain to be defined.

FIG. 1

Phylogenetic tree showing 16S rDNA sequences from porcine hindgut samples for low-G+C-content bacteria. The tree was constructed by neighbor-joining analysis of a distance matrix obtained from a multiple-sequence alignment. Bootstrap values (expressed as percentages of 100 replications) are shown at branch points: values of 90% or more were considered significant. Sequences derived from the database are shown in italics. The cultured isolates from the colonic wall are prefixed by asterisks. E. coli is used as the outgroup sequence. The scale bar represents substitutions per 100 nucleotides.

FIG. 2

Phylogenetic tree showing 16S rDNA sequences from porcine hindgut samples, for Streptococcus- and Lactobacillus-related sequences. The tree was constructed by neighbor-joining analysis of a distance matrix obtained from a multiple-sequence alignment. Bootstrap values (expressed as percentages of 100 replications) are shown at branch points: values of 90% or more were considered significant. Sequences derived from the database are shown in italics. The cultured isolates from the colonic wall are prefixed by asterisks. E. coli is used as the outgroup sequence. The scale bar represents substitutions per 100 nucleotides.

FIG. 3

PCR-RFLP profiles of Bacteroides and Prevotella strains from different sites in the pig hindgut digested with restriction endonuclease HhaI. Regions of extracted DNA are denoted as colonic (Co) lumen (L), wall (W), and wall surface (S) and cecal (Ca) lumen. The reference strains are as follows: lane 1, Prevotella ruminicola 23; lane 2, Prevotella albensis M384; lane 3, Bacteroides uniformis 1100; lane 4, Bacteroides thetaiotaomicron 5482. Some residual uncut material is present in lane 4.